Materials Science and Engineering C 24 (2004) 113 – 115
www.elsevier.com/locate/msec
Modification of electrode surface for covalent immobilization of laccase
De Quan, Woonsup Shin *
Department of Chemistry, Sogang University, 1 Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea
Abstract
Various methods for covalent immobilization of laccase on electrode surface were tested. In case of glassy carbon (GC) electrode, the
surface was modified by electrochemical oxidation of 1,5-pentanediol or by direct electrochemical oxidation of the electrode itself to
introduce carboxylic functional group. The peptide coupling between laccase and the functional groups of the modified electrode was done
by the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). The direct
modification method introduces denser carboxylic functional group. In the case of platinum (Pt) electrode, the surface was modified by
direct electrochemical oxidation to introduce hydroxy functional group, which was followed by coupling the enzyme with cyanuric chloride
(CC). Another method tested was the modification of the surface by silanization with 3-aminopropyltriethoxysilane (APTES), which was
followed by coupling the enzyme with glutaraldehyde (GA). Among the above four types of modification, the silanization method is the most
effective with respect to long-term stability and fast response for biosensor uses.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Modification of electrode surface; Covalent immobilization; Laccase; Biosensor
1. Introduction
Laccase ( p-diphenol:dioxygen oxidoreductase, EC 1.10.
3.2) is a blue multi-copper-containing enzyme, which catalyzes the oxidation of a variety of organic substrates coupled
to the reduction of molecular oxygen to water [1,2]. It
displays a broad specificity for the reducing substrates,
catalyzing the oxidation of different phenols, amino phenols
and aromatic diamines, etc. Laccases from various sources
have been successfully immobilized on various supports [3].
Recently, we reported laccase from DeniLitek could be
successfully immobilized on a Pt surface and be used to
detect phenolic compounds [4,5]. In this study, several
modification methods on GC and Pt surfaces were tested
and compared for biosensor uses.
isolated and purified from a commercial product from
DeniLitek from Novo Nordisk Co. according to the published methods [4,6]. Introduction of carboxyilic groups to
the GC electrode was performed by direct electrochemical
oxidation of the surface [7,8] or by electrochemical oxidation of 1,5-pentanediol [9]. Covalent immobilization of pphenylenediamine (PPD) or laccase on the modified GC
surface was done by use of EDC/NHS [10,11]. In the case of
laccase, GA vapor was used for cross-linking of the enzyme
with each other [12]. Pt electrode was modified either by
treatment with CC [13,14] or by silanization method [4].
Laccase was immobilized by the similar method used for
GC electrode.
3. Results and discussion
2. Experimental
3.1. Modification of electrode surface
Pt and GC disk working (4 mm in diameter), Pt wire
counter (spiral), and Ag/AgCl reference electrodes were
used for electrochemical measurements. BAS 50 W or
cDAQ-1604 (Elbio, Korea) potentiostat was used to run
CVs and measure current– time responses. Laccase was
GC surface was introduced carboxylic groups either by
direct oxidation of electrode itself or by oxidation of 1,5pentanediol. The amount of carboxylic group on the surface
was determined by detecting the amount of PPD that can be
connected to the modified GC surface easily by EDC/NHS
coupling. The surface coverages of PPD resulted from
different methods were calculated from CVs (Fig. 1 for
examples of direct oxidation and 1,5-pentanediol oxidation)
* Corresponding author. Tel.: +82-2-705-8451; fax: +82-2-701-0967.
E-mail address: [email protected] (W. Shin).
0928-4931/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2003.09.036
114
D. Quan, W. Shin / Materials Science and Engineering C 24 (2004) 113–115
oxidation peak currents of immobilized PPD (data not
shown) shows that the oxidation peak currents increase
linearly with scan rate up to 50 mV/s with a correlation
coefficient r = 0.995, as expected for a surface immobilized
reversibly oxidized/reduced redox couple [15]. PPD was also
immobilized on Pt surface by use of CC after introducing
Fig. 1. CVs with PPD immobilized GC electrode in 0.1 M phosphate buffer,
pH 7.0. (a) modification by direct oxidation at 2.7 V for 60 s; (b)
modification by 1,5-pentanediol oxidation. Scan rate: 2 mV/s, under Ar.
obtained at slow scan rate (2 mV/s) and shown in Table 1. The
direct modification gives denser carboxyl functional groups
than those by oxidation of 1,5-pentanediol. For example, the
surface coverage resulted from 1,5-pentanediol oxidation is
110 9 mol/cm2, which is only 5% of that from direct
oxidation at 2.7 V for 60 s. It has been suggested that the
resulted surface on a GC electrode anodized in an alkanol is
not as densely packed as that on a gold electrode modified by
self-assembly technique [9]. The scan rate dependences of the
Table 1
Comparison of surface coverages resulted from different modification
methods
Surface coverage C
(mol/cm2)*
Cyanuric chloride
1,5-Pentanediol
0.5 M Na2SO4, 2.3 V
0.1 M PBS, pH 6.8, 2.5 V
0.1 M PBS, pH 6.8, 2.7 V
* An average of three detections.
210
110
510
110
210
10
9
9
8
8
Fig. 2. Amperometric sensor signals of laccase immobilized electrode in
0.05 M phosphate buffer, pH 6.0. (a) direct oxidation at 2.7 V for 60 s, PPD
per injection: 5 AM; (b) 1,5-pentanediol oxidation, PPD per injection: 5
AM; (c) silanization, PPD per injection: 1 AM. Applied potential: +85 mV
vs. Ag/AgCl.
D. Quan, W. Shin / Materials Science and Engineering C 24 (2004) 113–115
115
hydroxy group on the surface. The surface coverage of PPD is
only 210 10 mol/cm2. Silanization with APTES was also
tried according to the published method [4].
resulted from the flexibility and the higher surface density of
the linker, 3-aminopropyltriethoxysilane.
3.2. Laccase immobilized electrode used as a biosensor
4. Conclusions
Laccase was immobilized by applying 5 Al of 6 mg/ml
enzyme solution in 0.1 M, pH 6.0 MES buffer on the EDC/
NHS-treated GC electrodes and exposing to the vapor of
25% aqueous GA solution. EDC-GA is a general method for
activation of surface carboxylic group and following crosslinking of an enzyme [10,12]. The enzyme modified electrode as a biosensor was tested by detecting PPD which is a
good substrate for laccase. The working potential of the
laccase electrode was fixed at 100 mV negative than the
reduction peak potential of the enzymatically oxidized form
of PPD, then the substrate was re-reduced on the electrode
surface, which given a reduction current (IR) in amperometric experiment.
Although the surface coverage of the directly oxidized
GC electrode is larger than that by oxidation of 1,5pentanediol, the response time (t90%) for the amperometric
detection of PPD is as long as 60 s (sensitivity=147 nA/AM,
Fig. 2a). The high degree of roughness caused by relatively
high potential oxidation of the electrode might be responsible for it. Contrary to this, response time of the laccase
immobilized on 1,5-petanediol oxidized GC electrode is
very fast, t90% is only 2 s (Fig. 2b). For the laccase electrode
immobilized via entrapment in polymer, the response time
was over 150 s [16]. The sensitivity of the laccase immobilized on 1,5-petanediol oxidized GC electrode is rather
small, 5 nA/AM, with PPD as the substrate. As mentioned
above, the low surface concentration of the carboxylic
functional groups should be responsible for it.
The sensor signal of the laccase covalently immobilized
Pt electrode via silanization is very stable and rather
reproducible (Fig. 2c). The sensitivity of the sensor is 350
nA/AM, which is comparable or superior to those reported
up to now [4,5,16,17]. The response time is very fast
(t90%<2 s), and the immobilized laccase can tolerate over
60 times successive injections of PPD (1 AM of per
injection) in the same detection, which indicates relatively
more loading of the enzyme. The stability of the sensor is
outstanding, which is as long as 2 months (retaining 80% of
initial activity) [4,5]. It can be reasonably concluded that
this sensor can totally tolerate at least 1000 times repeated
injection of 1 AM of PPD. The detection limit is about 40
nM of PPD (S/N=3), and the linear range is 0.15f30 AM.
Therefore, the laccase electrode can undoubtedly be used as
a biosensor for detection of diphenols. The reasons for most
performance of the sensor prepared by silanization compared to those by immobilization on GC electrode might be
Modification of GC electrode by direct electrochemical
oxidation can provide denser surface carboxylic functional
group than that by 1,5-pentanediol oxidation. Silanization
and following GA treatment is the most effective for
covalent immobilization of laccase for biosensor uses.
Acknowledgements
The authors acknowledge the financial support from
the Korea Research Foundation (KRF) by grants 2001042-G 00015, and the Ministry of Information and
Communication of Korea by grants from the contribution
of Advanced Backbone IT Technology Development
Project (IMT 2000-B3-2).
References
[1] B. Reinhammar, in: L. Rene (Ed.), Copper Proteins & Copper Enzymes, CRC Press Inc., Boca Raton, FL, 1984, chap. 1.
[2] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chem. Rev. 96
(1996) 2563 – 2605.
[3] N. Duran, M.A. Rosa, A. D’Annibale, L. Gianfreda, Enzyme Microb.
Technol. 31 (2002) 907 – 931.
[4] D. Quan, Y. Kim, K. Yoon, W. Shin, Bull. Korean Chem. Soc. 23 (3)
(2002) 385 – 390.
[5] D. Quan, Y. Kim, W. Shin, J. Electroanal. Chem. (2003) (in press).
[6] Y. Kim, N. Cho, T. Eom, W. Shin, Bull. Korean Chem. Soc. 23 (2002)
985 – 989.
[7] T. Atoguchi, A. Aramata, A. Kazusaka, M. Enyo, J. Electroanal.
Chem. 318 (1991) 309 – 320.
[8] A.D. Jannakoudakis, P.D. Jannakoudakis, E. Theodoridou, J. Appl.
Electrochem. 20 (1990) 619 – 624.
[9] H. Maeda, M. Itami, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 44
(12) (1996) 2294 – 2299.
[10] www.piercenet.com, Instructions for EDC, NHS.
[11] V.M. Mirsky, M. Riepl, O.S. Wolfbeis, Biosens. Bioelectron. 12
(9 – 10) (1997) 977 – 989.
[12] R.S. Freire, N. Duran, L.T. Kubota, Talanta 54 (2001) 681 – 686.
[13] G.R. Gustafson, C.M. Baldino, M.-M.E. O’Donnell, A. Sheldon, R.J.
Tarsa, C.J. Verni, D.L. Coffen, Tetrahedron 54 (1998) 4051 – 4065.
[14] C. Kempter, W. Potter, N. Binding, H. Klaning, U. Witting, U. Karst,
Anal. Chim. Acta 410 (2000) 47 – 64.
[15] R.W. Murray, in: A.J. Bard (Ed.), Chemically Modified Electrodes,
Marcel Dekker, New York, 1984, pp. 191 – 368.
[16] F. Lisdat, U. Wollenberger, A. Markower, H. Hortnagl, D. Pfeiffer,
F.W. Scheller, Biosens. Bioelectron. 12 (12) (1997) 1199 – 1211.
[17] T. Wasa, K. Akimoto, T. Yao, S. Murao, Nippon Kagaku Kaishi 9
(1984) 1398 – 1403.